Tin diiododeuteroporphyrin and therapeutic use thereof

ABSTRACT

Use of the novel compound tin diiododeuteroporphyrin and compositions containing it to inhibit heme mebabolism in mammals, to control the rate of tryptophan metabolism in mammals, and to increase the rate at which heme and iron are excreted by mammals.

BACKGROUND OF THE INVENTION

This invention relates to the novel compound tin diiododeuteroporphyrin(SnI₂ DP), to therapeutically useful compositions containing it, and tothe use of the compound and the compositions in treating variousmetabolic afflictions of mammals, particularly humans.

Heme is a red pigment comprised of four subunits called pyrroles; thesesubunits are chemically joined to form a single large tetrapyrrole(porphyrin) ring structure. A metal atom is chelated at the center ofthis porphyrin. In higher organisms this metal is iron and theporphyrin-iron ring structure is called protoporphyrin IX or heme. Inphysiological systems heme is bound to certain proteins; these hemeproteins bind oxygen at the site of the iron atom or they function ascomponents of membrane bound electron transport systems. Cellularrespiration, energy generation and chemical oxidations are dependent onthese heme proteins.

In mammals and other vertebrates heme is oxidatively degraded by anenzyme called heme oxygenase to form the open chain tetrapyrrolebiliverdin. Biliverdin is then reduced to bilirubin by another enzymebiliverdin reductase. In liver bilirubin is converted to mono- anddi-glucuronide conjugates by the hepatic glucuronyl transferase systemprior to its excretion.

Bilirubin is a toxic compound, but normally this toxicity is notmanifest since bilirubin is rapidly bound to plasma proteins,transported to liver, conjugated and execreted. However in the newborn,undesirably high concentrations of bilirubin exist in serum and mayproduce neurotoxicity. The intractable neurological syndrome known as"kernicterus" is the most severe manifestation of bilirubin toxicity.

The basis of this neonatal hyperbilirubinemia lies in a number offactors, mainly the rapid hemolysis of fetal erythocytes after birth anda developmental immaturity of the hepatic conjugating system whichnormally facilitates the excretion of bilirubin via the bile. The levelsof heme oxygenase, the rate limiting enzyme in the catabolism of heme tobilirubin are also markedly elevated at this time resulting in highrates of production of this bile pigment. Current methodologies forsuppressing severe neonatal jaundice include a. stimulation of thehepatic conjugating system for bilirubin by drugs, e.g. phenobarbital,b. partial or total exchange transfusion, and c. phototherapy. None ofthese methods is fully satisfactory since there are as yet manyunanswered questions with respect to their safety. In addition all thesemethods are directed towards the disposition of bilirubin after it hasbeen formed in the heme degradative sequence.

Elevated levels of bilirubin also often appear in the serum ofindividuals with diseases such as congenital anemias, thalassemia andsickle cell anemias as well as various forms of liver disease. Theconcentration of bilirubin in the serum of such individuals rarelyreaches the high levels found in neonates. It does, however, attainlevels which may be toxic and should be controlled.

It is therefore desirable to have available methods and materials toinhibit the catabolism of heme in order to prevent the accumulation ofbilirubin in serum.

Copending and commonly assigned U.S. patent application Ser. No.684,169, now abandoned, describes the use of tin protoporphyrin IX(SnPP) in the treatment of elevated levels of bilirubin in neonates andadults. Copending and commonly assigned U.S. patent application Ser. No.715,515 describes the use of tin mesoporphyrin (SnMP) for the samepurpose.

Maintenance of a proper equilibrium or balance of tissue heme content isessential to the normal physiological functioning of cells. When thisequilibrium is disturbed by any condition characterized by excess hemedegradation to bilirubin--as exemplified by the circumstances listedabove--it would be clinically valuable to have a pharmacologicalmechanism for restoring the equilibrium state of heme in cells byfacilitating the excretion of the excess amount of heme from the body.

In association with but independent of the conditions described above,excess iron also accumulates in the body and this accumulation of themetal over time can produce deleterious and even lethal consequences forthe host. This excess of iron may derive from several sources; e.g.cooking methods (iron pots) or directly via the diet (e.g.,iron-overload induced cutaneous porphyria), from excess therapeuticadministration of the metal in an attempt vigorously to treatunresponsive anemias; from hypertransfusions to which certain patientswith blood disorders are subject; idiopathically from the genetic andacquired disorders collectively known as "hemachromatosis"; from certainindustrial exposures; but the most common causes of excess irondeposition in tissues, and the resultant pathologic consequences whichderive thereof, are a consequence of common congenital hemolytic anemiassuch as sickle cell disease, the various forms of thalassemia, G-6-PDdeficiency, hereditary spherocytosis and the like. In these disorders, agreatly shortened red cell life span results in continuous largedepositions of iron in tissues to an extent exceeding the capacity ofthe body to re-utilize the metal. These tissue concentrations of ironrise to very high, toxic levels and lead to impairment of vital organfunctions manifest, for example, by cardiomyopathy, pancreaticinsufficiency (diabetes) and generalized endocrine failure.

There is no physiological mechanism for excreting this excess of ironand the only generally available therapeutic modality for this purposeis a pharmacological agent known as desferrioxamine. This agent is notspecific for iron however and chelates other metals as well. It must, inorder to be reasonably effective, be given intramuscularly and causessubstantial local inflammation at the site of injection. Further,original suggestions that it was non-toxic have proved incorrect, and alarge number of toxic reactions in treated patients have now beenreported to occur after its use.

SnPP and SnMP as described in the copending and commonly assignedapplications identified above both manifest the extremely advantageousproperties of greatly enhancing the biliary excretion of iron into theintestinal contents where the metal is eliminated. SnPP and SnMP act inthis additional fashion by blocking the binding of heme to hemeoxygenase, thus preventing the release of iron which normally occurs inthe process of heme catabolism and allowing one atom of iron to beexcreted into the intestine with every molecule of uncatabolized heme.

Tryptophan is an essential amino acid which has profound effects on anumber of metabolic pathways in the whole animal, including man,particularly in the nervous system. Tryptophan is metabolizedprincipally in the liver. Tryptophan which is not metabolized in theliver accumulates in the plasma and in the brain. Brain levels oftryptophan are dependent on plasma levels of the amino acid which inturn are regulated by liver tryptophan pyrrolase. Tryptophan in thebrain is metabolized by a different route than in the liver. One of theprincipal metabolic products of tryptophan in the brain is5-hydroxytryptamine, or serotonin. The concentrations of tryptophan andserotonin in the brain are closely regulated in humans. Increasedconcentration of these products are associated with hepaticencephalopathy and migraine headaches. Encephalopathy is a knownaffliction characterized by degenerative changes in the brain cellsleading to confused states and other abnormal behaviour patterns as wellas convulsions, stupor and coma. Decreased concentrations of theseproducts have been implicated in narcolepsy, depression and nyoclonicdisorders characterized by uncontrolled jerky movements.

Tryptophan pyrrolase is a heme dependent enzyme which occurs in theliver of humans. It catalyzes the oxidative cleavage of tryptophan toN-formylkynurenine and is the first and rate-limiting enzyme in thecatabolism of tryptophan in the liver. The active holoenzyme is normallyabout 50% saturated with heme, but fluctuations in the availability ofcellular heme produce rapid changes in the enzyme activity by convertingthe inactive, heme-free apoenzyme to the active heme containingholoenzyme.

More specifically, an increase in the amount of heme in the liver suchas can be produced by parenteral administration of SnPP or SnMP as aresult of the ability of these compounds to block the catabolism of hemecauses increased saturation of tryptophan pyrrolase as the active formof the enzyme. The increased activity of the enzyme resulting from itsincreased saturation with heme causes an increased rate of tryptophanmetabolism in the liver. As a result there is less spill-over of intacttryptophan into the plasma and, ultimately, less accumulation oftryptophan and serotonin in the brain.

SnPP and SnMP, as will be apparent from the above, are very usefuladditions to the medical armamentarium. However, they both have thedisadvantage that they are photosensitizing agents. When the therapeuticagent is administered it spreads throughout the body and, because, itabsorbs light i.e. sunlight or light from ordinary fluorescent bulbs,causes skin rashes, flushed skin and general discomfort. It is,therefore, of significant medical interest to find agents which have theadvantages of SnPP and SnMP without the disadvantage of their propensityto photosize when exposed to light in the long wave length ultravioletregion.

THE INVENTION

It has now been discovered that the novel compound tindiiododeuteroporphyrin (SnI₂ DP) can be employed in the treatment ofmammals including humans in need of such treatment to decrease the rateof heme metabolism, to increase the rate at which heme is excreted andto control the rate of tryptophan metabolism in the liver.

SnI₂ DP is a novel compound which may be prepared by refluxingdiiododeuteroporphyrin in acetic acid solution with tin acetate. Theprocedure is as follows:

In a round bottom flask, 37.5 milligrams of tin acetate (Alfa Chemicals)was dissolved in 3.75 ml glacial acetic acid (1% solution) and wasslowly brought to a boil on a hot plate by refluxing with a condenser. Asolution of 25 mg diiododeuteroporphyrin dissolved in 0.6 ml pyridineand 0.75 ml chloroform was added to the boiling tin solution withconstant stirring. Reaction commenced with the addition ofdiiododeuteroporphyrin. Aliquots of 10 microliters of the reactionmixture were removed and added to pyridine to follow spectral changes.The original starting mixture was a cloudy, brownish suspension. After24 hours of heating, the mixture had taken on a clear crimsonappearance. The reaction was continued until the four line spectrum hadbeen converted to a two line spectrum (reaction time 47 hours). Thereaction mixture was allowed to cool overnight and N hydrochloric acidwas added with constant stirring which was continued for one hour. Thesuspension was filtered, washed with water and air dried to obtain thedesired product.

Several studies have been made to establish the efficacy of SnI₂ DP forthe purposes aforesaid.

In an initial study using rat spleen microsomes as the source of hemeoxygenase the ability of SnI₂ DP to inhibit the activity of hemeoxygenase to convert heme to bilirubin was determined. The inhibitionconstant K_(i) was found to be 0.069±0.007 μM as determined by use ofLineweaver Burke plots and the formula:

    Km.sub.APP =Km/K.sub.i ×[I]+Km

where

I=SnI₂ DP concentration

Km=rate constant for heme oxygenase

Km_(APP) =rate constant for heme oxygenase in the presence of SnI₂ DP

Four separate determinations of K_(i) were made: 0.057, 0.088 0.062,0.070 Ave 0.069±0.007.

This inhibition constant is approximately the same as the constants forSnPP and SnMP.

The physical characteristics of SnI₂ DP are as follows:

(1) Absorption spectrum: SnI₂ DP (molecular weight 915) exhibits 3 peaksin the visible region of the spectrum when dissolved in pyridine. Theyare at 418 nm, 543.9 nm and 582.4 nm. The absorption spectrum of SnI₂ DPis similar to those of Sn-protoporphyrin (SnPP) and Sn-mesoporphyrin(SnMP).

(2) Fluorescence characteristics: A 1 μM solution of SnI₂ DP in pyridinewas excited at 400 nm and 2 emission peaks were detected at 578 nm and635 nm. The measurable fluorescence represents less than 5% of thatfound for SnPP and SnMP.

(3) Singlet and triplet lifetimes: The singlet lifetime of SnI₂ DP wasdetermined to be 23 psec (this is at the lower levels of instrumentdetection). The singlet lifetimes of SnPP and SnMP are 500-600 p sec.The triplet lifetime of SnI₂ DP was determined to be 0.2 millisec. Thetriplet lifetime sof SnPP and SnMP are 0.5 and 2.5 millisecondsrespectively. Thus substitution at positions C₂ and C₄ of theprotoporphyrin macrocycle significantly influences the triplet lifetime,i.e., C₂ H₅ :C₂ H₄ :I₂ →2.5:0.5:0.2 millisec.

(4) Thin layer chromatography in 85:15:1.3 benzene/methanol/formic acidwith the compound dissolved in pyridine resulted in an R_(f) value of0.16.

It will be seen that SnI₂ DP is a synthetic tin metalloporphyrin inwhich, though iodine substitution at C₂ and C₄ of the deuteroporphyrinmacrocycle, the fluorescence is quenched by 95% by comparison with SnPP.In addition the triplet lifetime of SnI₂ DP is 0.2 milliseconds comparedwith SnPP and SnMP in which the triplet lifetimes are 0.5 and 2.5milliseconds respectively. This indicates that SnI₂ DP has lessphotosensitizing potential than SnPP and SnMP.

The in vivo ability of SnI₂ DP to reduce serum bilirubin was establishedby studies in neonatal rats. The studies were conducted with fourseparate litters each containing ten neonates.

To prepare the solution for parenteral administration, SnI₂ DP was takenup in a small volume of 0.2N sodium hydroxide, adjusted to pH 7.4 with1N hydrochloric acid and made up to final volume with 0.9% sodiumchloride. The solution as prepared and used contained a final SnI₂ DPconcentration of 10 μmol/kg body weight in each 0.1 ml injection volume.Control neonates received 0.1 ml of 0.9% sodium chloride at birth. Onegroup of animals was sacrificed at 24 hours which is normally the peakof bilirubin for neonate rates. The total bilirubin in serum wasestimated by the method of Roth, Clin. Chem. Acta, 17 487-492, 1967. Theresults are shown in Table I.

                  TABLE I                                                         ______________________________________                                        Effects of SnI.sub.2 DP on tissue heme oxygenase activity                     and serum bilirubin levels in newborn rats.                                   Heme Oxygenase                                                                (nmol/mg p h)            Serum Bilirubin                                      Treatment                                                                             Liver      Kidney.sup.xx                                                                          Spleen.sup.xx                                                                        (mg/dl)                                    ______________________________________                                        Saline  9.44 ± 0.24                                                                           2.81     6.76   0.53 ± 0.06                             SnI.sub.2 DP                                                                          .sup. 8.07 ± 0.06.sup.x                                                               1.13     3.74   .sup. 0.34 ± 0.02.sup.x                 ______________________________________                                         .sup.x p < 0.05 when compared to control values                               .sup.xx Kidney and spleen bulked                                         

The test was repeated and all parameters measured were similar 48 hoursafter birth except kidney heme oxygenase activity which was lower inSnI₂ DP treated neonates (0.88 nmol/mg p h) when compared to controlanimals (2.14 nmol/mg p h).

It will be apparent from the table that parenteral administration ofSnI₂ DP to rat neonates prevented the immediate and significant increasein the levels of serum bilirubin that normally occurs in the animals 24hours after birth.

The activity in this respect is roughly the same as SnPP and about 10%that of SnMP. The activity in rats for SnPP has been found to be totallypredictive of the activity in humans.

The next study established the ability of SnI₂ DP to inhibit the abilityof α-aminolevulinic acid (ALA) to produce jaundice. This study of theefficacy of SnI₂ DP to control hyperbilirubinemia thereby preventing thejaundice which develops in rats treated 7 days after birth with ALA isdescribed by Drummond and Kappas, J. Clin. Invest. 74, 142-149 (1984).In the test, ALA (50 μmol/100 g body weight) is administered to suckling7 day old rats at 0,4 and 8 hours, the animals sacrificed 16 hours afterthe last administration of ALA, and the serum biliribin measured. Theresults are shown in Table II.

The capacity of SnI₂ DP to control the increase of serum bilirubin inALA treated neonatal rats is apparent. The results are directlypredictive of the results expected with humans, both as neonates and asadults.

The table also shows the ability of SnI₂ DP to prevent any increase inhepatic heme oxygenase activity associated with ALA administration. Hemeoxygenase was determined as described by Drummond and Kappas Proc. Natl.Acad. Sci. USA 78:6466(1981).

                  TABLE II                                                        ______________________________________                                        Effect of SnI.sub.2 DP on hepatic heme oxygenase activity                     and serum bilirubin levels on ALA-treated hyperbilirubinemia                  in 7-day old suckling rats                                                                Heme Oxygenase                                                                (nmol/mg p h)                                                                              Serum Bilirubin                                      Treatment   Liver        (mg/dl)                                              ______________________________________                                        Saline      5.77 ± 0.13                                                                             0.32 ± 0.02                                       ALA         11.29 ± 0.33                                                                            0.99 ± 0.06                                       SnI.sub.2 DP                                                                              .sup. 8.29 ± 0.48.sup.x                                                                 .sup. 0.54 ± 0.02.sup.x                           ______________________________________                                         .sup.x p < 0.01 when compared to ALA treated animals.                    

In the next in vivo test designed to measure the decrease in bilirubinproduction, SnI₂ DP was administered intravenously to five adult rats,12-15 weeks old at a dose of 10 μmol/kg body weight. Five control ratswere administered saline. The bile duct was cannulated and the bilefluid analyzed for bilirubin content. The results are shown in TableIII.

                  TABLE III                                                       ______________________________________                                        Drop In Biliary Bilirubin Production                                          After SnI.sub.2 DP Administration                                                       SnI.sub.2 DP                                                                              Control   Difference                                    Experiment                                                                              % decrease  % decrease                                                                              %                                             ______________________________________                                        1         47          38         9                                            2         44          26        18                                            3         48          30        18                                            4         60          26        34                                            5         35          8.6       26                                            ______________________________________                                    

The average difference between treated and control decline in biliarybilirubin was 21%. This difference was significant (p<0.01). This dropis similar to that (˜25%) previously reported for SnPP. Simionatto etal. J. Clin. Invest. 75:513:(1985).

In addition, the activity of hepatic, renal and splenic heme oxygenasewas decreased in bile duct cannulated animals administered SnI₂ DP.

The results which confirm these data are shown in Tables III and IV.

                  TABLE IV                                                        ______________________________________                                        Effect of SnI.sub.2 DP on tissue heme oxygenase activity                             Heme Oxygenase (nmol/mg p h)                                           Treatment                                                                              Liver       Kidney     Spleen                                        ______________________________________                                        Saline   4.03 ± 0.24                                                                            1.74 ± 0.29                                                                           9.53 ± 0.95                                SnI.sub.2 DP                                                                           .sup. 2.06 ± 0.75.sup.x                                                                .sup. 0.45 ± 0.03.sup.x                                                               .sup.  5.37 ± 1.09.sup.xx                  ______________________________________                                         SnI.sub.2 DP administered i.v. at a dose of 10 μmol/kg b.w.                .sup.x p < 0.01                                                               .sup.xx p < 0.05                                                         

When compared to saline treated controls, n=5.

From these results it is seen that SnI₂ DP is as effective as SnPP inlowering bilirubin in bile (21% and about 25%, respectively) when thesame does (10 μmol/kg) body weight was administered. Thus, SnI₂ DPappears to be similar to SnPP, and not as effective as SnMP in itsability to lower heme oxygenase activity, lower serum and bilirubinlevels in bile.

These findings are especially important with adults under chronictreatment for any of the maladies described above. They are alsoimportant for neonates and infants who may need multiple doses of themetalloporphyrin to control persistent jaundice.

Simultaneous intraveous (IV) administration of heme (6.1 μmol/kg b.w.)and SnI₂ DP was examined in four pairs of bile duct cannulated adultmale rats. The effect of simultaneous administration of heme and SnI₂ DPon heme levels in bile and tissue heme oxygenase activity are shown inTables V and VI respectively.

                  TABLE V                                                         ______________________________________                                        Effect of SnI.sub.2 DP on heme excretion in bile                                          Heme excreted in bile                                                         (% of administered dose)                                          Experiment    SnI.sub.2 DP                                                                             Control                                              ______________________________________                                        1             51.8       10.8                                                 2             48.8       35.4                                                 3             48.2       27.3                                                 4             30.7        18.10                                               Average       44.9 ± 4.79*                                                                          22.9 ± 5.36                                       ______________________________________                                         *p < 0.05 when compared to control administered heme.                    

SnI₂ DP administration resulted in a significant increase in the amountof heme excreted in bile of bile duct cannulated rats. The levels oftissue heme oxygenase were also decreased in SnI₂ DP treated animals(Table VI).

                  TABLE VI                                                        ______________________________________                                        Effect of SnI.sub.2 DP on tissue heme oxygenase activity                      after simultaneous administration of heme                                             Heme Oxygenase (nmol/mg p h)                                          Treatment Liver       Kidney    Spleen                                        ______________________________________                                        Saline    8.15 ± 1.64                                                                            1.45 ± 0.27                                                                          11.18 ± 1.41                               SnI.sub.2 DP                                                                            .sup. 5.03 ± 0.72.sup.x                                                                1.04 ± 0.22                                                                          8.15 ± 0.52                                ______________________________________                                         .sup.x p < 0.01 compared to saline treated animals (n = 4)               

Each group was administered heme (6.1 μmol/kg b.w.) simultaneously witheither SnI₂ DP or saline.

With young mammals, the therapeutic compositions of this invention willbe administered at a dosage of from 0.5 to 25 mg/kg of body weight.While appreciable variations from this range can be tolerated withoutunacceptable adverse effects, this range appears to be the mostpractical. Any of the usual parenteral routes may be employed. Normally,one injection will suffice to maintain the bilirubin concentration at adesired low level until the infant reaches the age when the metabolismof heme is in balance. It is preferred, however, to monitor the serumbilirubin concentration and to utilize a booster dose, if necessary.

With adults afflicted with sickle cell anemia or another conditionresulting in a constantly increased bilirubin concentration, the dosageunit is normally smaller since in all but the most acute situations, thebilirubin concentration is not as high as in infants. The standarddosage with adults will normally be from about 0.5 to 5 mg/kg of bodyweight administered in repeated doses.

Therapeutic compositions of this invention will be prepared by the usualprocedures employed for such purposes. The usual pharmaceutical carriersfor parenteral administration may be used such as aqueous media madeisotonic by the addition of sodium chloride, glucose or other standardsolutes. Typically the compositions will be buffered, for example with aphosphate buffer to a pH of about 7 to 8, preferably 7.4 to 7.5. Theconcentration of SnI₂ DP in the composition will be from 2 to 25g/liter, so that they can be formed into dosage unit forms adequate toprovide a dosage of from 2 to 25 mg/kg body weight. Accordingly, thedosage units will normally contain from 2 μmol/ml to 25 μmol/ml ofsolution.

What is claimed is:
 1. Tin diiododeuteroporphyrin.
 2. A pharmaceutical composition for parenteral administration comprising tin diiododeuteroporphyrin and pharmaceutically acceptable carrier.
 3. A pharmaceutical composition in dosage unit form for parenteral administration comprising tin diiododeuteroporphyrin at a concentration of 0.5 μmol/ml to 25 μmol/ml in a pharmaceutically acceptable carrier.
 4. A method of decreasing the rate of metabolism of heme in mammals in need of such treatment by administering to the mammal an amount of tin diiododeuteroporphyrin which is effective to decrease said rate.
 5. A method as in claim 4 wherein the mammal is a human. 